1. Field of the Invention
This invention relates to methods and apparatus for the imaging of marked materials associated with the surface of a substrate. In particular, the invention relates to methods and apparatus for imaging arrays of polymer sequences such as oligonucleotide arrays.
Biological assays involving fluorescent label molecules or scattering structures to detect, quantify or identify target chemical species bound to surfaces use optical detection and imaging systems. Arrays of different chemical probe species provide methods of highly parallel detection, and hence improved speed and efficiency, in assays. These arrays are sometimes referred to as chip or microarray technologies.
One method for detecting nucleic acids is to employ nucleic acid probes that have sequences complementary to the target nucleic acid sequences. A nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support. The detection of its signal depends upon the nature of the label or reporter group. Usually, the probe is comprised of natural nucleotides, such as ribonucleotides and deoxyribonucleotides and their derivatives, although unnatural nucleotide mimetics such as 2xe2x80x2-modified nucleosides, peptide nucleic acids and oligomeric nucleoside phosphonates are also used. Commonly, binding of the probes to the target is detected by means of a label incorporated into the probe. Alternatively, the probe may be unlabeled and the target nucleic acid labeled. Binding can be detected by separating the bound probe or target from the free probe or target and detecting the label. In one approach, a sandwich is formed comprised of one probe, which may be labeled, the target and a probe that is or can become bound to a surface. Alternatively, binding can be detected by a change in the signal-producing properties of the label upon binding, such as a change in the emission efficiency of a fluorescent or chemiluminescent label. This permits detection to be carried out without a separation step. Finally, binding can be detected by labeling the target, allowing the target to hybridize to a surface-bound probe, washing away the unbound target and detecting the labeled target that remains.
Direct detection of labeled target nucleic acid hybridized to surface-bound polynucleotide probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations.
In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective attachment to matching probe sites takes place. The array is then washed and imaged so as to reveal for analysis and interpretation the sites where attachment occurred.
These chip technologies, such as DNA arrays and protein matrix arrays, need to be scanned to measure the number densities of labeled molecules and hence the concentration of target (or probe) molecules in solution. This sensing process is accomplished by means of a fluorescence imaging system. In order to reduce the deleterious effects of background from either the slide substrate (such as the glass slide) or the solution (assuming a wet-scanning system), confocal scanning systems are employed resulting in increased performance.
Confocal microscopes generally employ a pinhole that is confocal with an illuminated spot on a specimen to reject light that is not reflected or emitted from objects in the focal plane. This rejection of out-of-focus light enables the microscope to collect and combine a series of optical slices at different focus positions to generate a two or three-dimensional representation of the specimen. However, confocal microscopes tend to be complex devices with many moving parts. These moving parts often involve expensive high-precision stages necessitated by high-resolution scanning. A scanner with such high-precision stages will typically cost tens, and even hundreds, of thousands of dollars.
Optical scanning imaging techniques are employed in devices such as scanning laser microscopes, confocal scanning laser microscopes, tandem scanning confocal microscopes, scanning laser ophthalmoscopes and flying spot television devices. Confocal imaging systems can provide enhancements in contrast and in dynamic range. Certain of these imaging systems include moving optical elements for deflecting a laser beam so that an illumination spot is swept across the object to be scanned. Other such systems employ mechanical elements to rotate and illuminated pinhole for the same purpose. In the tandem scanning confocal microscopes a plurality of illumination spots is moved simultaneously to provide source multiplexing, necessary because the source does not have the higher radiance of a laser.
There is a need for a simple high-resolution scanner with a reduced number of moving parts. Ideally, the number of moving parts is one or fewer and the motion of any moving parts is simple.
2. Brief Description of Related Art
Webb, et al., (U.S. Pat. No. 5,563,710) discuss an imaging system with confocally self-detecting laser.
Stem, et al., (U.S. Pat. No. 5,631,734) discuss a method and apparatus for detection of fluorescently labeled materials.
Apparatus and method with tiled light source array for integrated array sensing is described in U.S. Pat. No. 5,812,272 (King, et al.).
U.S. Pat. No. 5,900,949 (Sampas) discloses a CCD imager for confocal scanning microscopy.
An optical scanning apparatus is discussed by Dorsel, et al., in U.S. Pat. No. 5,585,639.
Systems and methods for detection of labeled materials are disclosed in WO 97/43611 (Stern).
One embodiment of the present invention is an imaging apparatus comprising a holder for a surface, a light source adapted to illuminate the surface, a diffractive element between the holder and the light source and an imaging detector adapted to receive light from the surface.
Another embodiment of the present invention is a method for imaging a surface comprising a plurality of discrete features. Light is selectively diffracted on to one or more predetermined features of the surface at a predetermined point in time. This step is repeated until substantially all of the features on the surface are illuminated. Light is detected from the surface to thereby image the surface.